The broad objective of this proposal is a better understanding of how metal-containing enzymes work. The more specific aims involve characterization of three classes of Fe-based metalloenzymes with 'unusual' active sites -- nitrogenase (N2ase), hydrogenase (H2ase), and methane monooxygenase (MMO). We also seek to understand how special Fe-S cluster ('Wbl') proteins from Mycobacteria and Streptomyces bacteria sense NO and O2 in their environment. The questions that we hope to answer revolve around molecular structure (what atoms are where) and dynamics (how the atoms move). To achieve this knowledge, we have arranged close collaborations between chemists, biochemists, and spectroscopists. We will develop or enhance spectroscopic 'probes' that allow us to answer questions about the structure of enzyme intermediates. These include x-ray and nuclear techniques at synchrotron radiation labs -- XAFS and NRVS. We will balance these 'large facility' methods with campus-based spectroscopies including EPR, FT-IR, and Mssbauer, both as direct probes and combined with UV-visible photolysis methods. For N2ase, H2ase, and MMO, the questions that we plan to address are: How does structure change during the course of the catalytic cycle? Where do substrates and inhibitors bind? What are the undefined light atoms? The same questions apply to the interactions of NO and O2 with Wbl proteins, except that the reactions involve sensing small molecules as opposed to catalysis. Our spectroscopic techniques will allow us to monitor the enzyme active sites. We will use this capability to focus on structural and dynamic issues that are beyond the reach of protein crystallography.